Home Genome Editing with CRISPR/Cas EnGen® SpRY Cas9

EnGen® SpRY Cas9
Gray property icon for NEB r3.1 recombinant Gray property icon for incubation temperature 37° Gray property icon for heat inactivation 80°

  • Eliminate sequence constraints for dsDNA targeting with non-specific PAM (5´-NNN-3´ PAM)
  • Digest large plasmids in cloning workflows successfully
  • Use in conjunction with the EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322), EnGen Mutation Detection Kit (NEB #E3321), and NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621)
Blog_ContextualAd
Catalog # Cat # Concentration Conc Size List Price List Price Your Price Quantity
M0669T 20 µM 500 pmol $179.00 $179.00
M0669M 20 µM 2,500 pmol $712.00 $712.00
Please enter a quantity for at least one size
Need a custom/large volume order? Contact us
  • EnGen SpRY Cas9 from Streptococcus pyogenes is an engineered, RNA-guided, DNA endonuclease that catalyzes site-specific cleavage of double-stranded DNA (dsDNA). Targeting requires a ~100 nucleotide single guide RNA (sgRNA) with complementarity to the 20-nucleotide region immediately upstream of a protospacer adjacent motif (PAM) on the dsDNA substrate. EnGen SpRY Cas9 encodes 11 point mutations (A61R, L1111R, D1135L, S1136W, G1218K, E1219Q, N1317R, A1322R, R1333P, R1335Q, T1337R) designed to diminish the requirement for a PAM. Unlike the canonical 5´-NGG-3´ PAM requirement of wild-type Spy Cas9, SpRY Cas9 has been demonstrated to have almost no PAM requirement in vitro, cleaving at many sites with a 5´-NNN-3´ PAM (although it exhibits a preference for 5´-NRN-3´ over 5´-NYN-3´ PAMs in vivo) (1,2). DNA cleavage by EnGen SpRY Cas9 produces a double-stranded break occurring 3 nucleotides upstream of the PAM. EnGen SpRY Cas9 contains Simian virus 40 (SV40) T antigen nuclear localization sequence (NLS) on the C-terminus of the protein.



    Figure 1: EnGen SpRY Cas9 has no sequence constraints for dsDNA targeting in vitro

    M0669 Mechanism

    EnGen SpRY Cas9 is a variant of Cas9 nuclease from S. pyogenes with several point mutations within the PAM interacting domain (1).  Unlike wildtype Cas9, EnGen SpRY Cas9 is not constrained by the presence of an NGG PAM and can produce a double stranded break three nucleotides upstream of any trinucleotide sequence in in vitro applications.




    Figure 2: Streamline large construct cloning workflows with EnGen SpRY Cas9 and NEBuilder HiFi DNA Assembly


    M0669 Cleavage Specificty

    Demonstration of EnGen® SpRY Cas9 cleavage flexibility. A) Schematic of pXba with restriction endonuclease BsrGI recognition sites denoted. The rectangular box contains the BsrGI recognition sequence with the cleavage sites denoted by red triangles. B) Sequences of three sgRNAs used to target BsrGI sites in pXba. Cleavage sites for both EnGen SpRY Cas9 and BsrGI are denoted by red triangles. SpRY Cas9 non-canonical PAMs are denoted in yellow text. C) 1 µg of pXba (22,563 bp) was digested with either 10 units of BsrGI or 1 µM EnGen SpRY Cas9 and 1 µM each of three sgRNAs targeting all three BsrGI sites present in pXba in 1X NEBuffer r3.1. All reactions were incubated at 37°C for 1 hour followed by 80°C for 5 minutes. The DNA banding pattern of the BsrGI and SpRY digests were compared by gel electrophoresis using the Agilent gDNA ScreenTape System on a 4200 TapeStation instrument. Cleavage of pXba by both BsrGI and SpRY Cas9 targeted to the BsrGI sites resulted in a nearly identical band pattern. Digestion of 1 µg of pXba with EnGen SpRY Cas9 and sgRNA concentrations as low as 50 nM resulted in a similar cleavage compared with 1 µM results. Undigested pXba was run as a control, however circular DNA does not run accurate to size in the gDNA ScreenTape System. A vertical grey line marks where two different ScreenTapes were used.



    Figure 3: Linearization of a 22.6 kb plasmid with EnGen SpRY Cas9 followed by cloning with NEBuilder HiFi DNA Assembly.


    M0669 Suitability for NEBuilder

    Determination of suitability of EnGen SpRY Cas9 digestion reactions for subsequent use in NEBuilder HiFi DNA Assembly reactions. A) Schematic depicting the linearization and cloning strategy. 1 µg of pXba (22,563 bp) was linearized downstream of the start codon of the pVII orf with 50 nM EnGen SpRY Cas9 and 50 nM sgRNA (targeting sequence of 5´-CTTTTTGAGCAAACATGTCC-3´) in 1X NEBuffer r3.1 for 1 hour at 37°C. The reactions were either spin column purified or left unpurified before proceeding to DNA assembly. The NEBuilder HiFi DNA Assembly kit was used to insert an oligonucleotide encoding a nuclear localization signal (NLS) tag to pVII according to the recommended protocol. B) Inspection of purified and unpurified linearized pXba after EnGen SpRY Cas9 digestion using the Agilent gDNA ScreenTape System. Undigested pXba was run as a control, however circular DNA does not run accurate to size in the gDNA ScreenTape System. C) Measurement of the number of colonies grown after transformation with DNA assembly reactions using either purified or unpurified EnGen SpRY Cas9 digests. A no insert control was included to qualitatively assess how many transformants arise from undigested plasmid following EnGen SpRY Cas9 digest. Purification of linearized pXba prior to DNA assembly reduced the percentage of background colonies. 8-9 colonies of transformants derived from purified and unpurified EnGen SpRY Cas9 digests were picked and checked for proper DNA assembly by PCR using primers spanning the insert. 100 percent of clones from both purified and unpurified EnGen SpRY Cas9 digests displayed the correct insert (data not shown).



    Product Source

    An E. coli strain that carries the cloned Cas9 gene from Streptococcus pyogenes with C-terminal Simian virus 40 (SV40) T antigen nuclear localization signal (NLS) and a C-terminal 6XHis tag
    This product is related to the following categories:
    CRISPR/Cas Nucleases

    • Reagents Supplied

      The following reagents are supplied with this product:

      NEB # Component Name Component # Stored at (°C) Amount Concentration

    • Properties & Usage

      Reaction Conditions

      1X NEBuffer™ r3.1
      Incubate at 37°C

      1X NEBuffer™ r3.1
      100 mM NaCl
      50 mM Tris-HCl
      10 mM MgCl2
      100 µg/ml Recombinant Albumin
      (pH 7.9 @ 25°C)

      Storage Buffer

      300 mM NaCl
      10 mM Tris-HCl
      0.1 mM EDTA
      1 mM DTT
      50% Glycerol
      pH 7.4 @ 25°C

      Heat Inactivation

      80°C for 5 minutes

    • Product Notes

      1. 20 µM is equal to 3.25 mg/ml

    • References

      1. Walton, et al. (2020). Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science. Apr 17;368(6488), 290-6. DOI: 10.1126/science.aba8853
      2. Christie, et al. (2023). Precise DNA cleavage using CRISPR-SpRYgests. Nat. Biotechnol.. Mar 41, 409-16. DOI: 10.1038/s41587-022-01492-y

  • Protocols, Manuals & Usage

  • Quality, Safety & Legal

    • Quality Control Assays

      Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

    • Certificate of Analysis

      The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

    • Safety Data Sheets

      The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    • Legal and Disclaimers

      Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

      This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

      New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Other Products You May Be Interested In

Ineligible item added to cart

Based on your Freezer Program type, you are trying to add a product to your cart that is either not allowed or not allowed with the existing contents of your cart. Please review and update your order accordingly. If you have any questions, please contact Customer Support at freezers@neb.com or 1-800-632-5227 x 8.

Choose your country

North America

Europe

Asia-Pacific

If you don't see your country above, please visit our international site

Session Expired

You have been idle for more than 20 minutes, for your security you have been logged out. Please sign back in to continue your session.

Institution Changed

Your profile has been mapped to an Institution, please sign back for your profile updates to be completed.

Sign in to your NEB account

To save your cart and view previous orders, sign in to your NEB account. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site.